Team:Cambridge/Protocols/PCRcolony

From 2012.igem.org

Parts for a reliable and field ready biosensing platform

Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field. We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

Judging Form

Please help the judges by filling out this form. Tell them what medal you think you deserve and why. Tell them which special prizes you should win. Help them find your best parts. Show them how you thought about the safety of your project. Helping the judges will help you too.

Team: Cambridge

Region: Europe

iGEM Year:2012

Track:Foundational Advance

Project Name:Parts for a reliable and field ready biosensing platform

Project Abstract: Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field.

We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

iGEM Medals for non-software teams

Because we met the following criteria (check all that apply and provide details where needed)

Requirements for a Bronze Medal

√Register the team, have a great summer, and plan to have fun at the Regional Jamboree.

√Successfully complete and submit this iGEM 2012 Judging form.

√Create and share a Description of the team's project using the iGEM wiki and the team's parts using the Registry of Standard Biological Parts.

√Plan to present a Poster and Talk at the iGEM Jamboree.

√Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological Parts. Including:

√Primary nucleaic acid sequence

√Description of function

√Authorship

Safety notes, if relevant.

√Acknowedgment of sources and references

√Submit DNA for at least one new BioBrick Part or Device to the Registry.

Additional Requirements for a Silver Medal

√Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device.

√Enter this information and other documentation on the part's 'Main Page' section of the Registry Part Number(s): BBa_K911004

Additional Requirements for a Gold Medal: (one OR more)

Improve an existing BioBrick Part or Device and enter this information back on the Experience Page of the Registry.Part Number(s): None

√Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.Link to this information on your wiki. Page name: Team:Cambridge/Outreach/Collaboration

iGEM Prizes

All teams are eligible for special prizes at the Jamborees. more... To help the judges, please indicate if you feel you should be evaluated for any of the following special prizes:

√Best Human Practice Advance

√Best Experimental Measurement

Best Model

Please explain briefly why you should receive any of these special prizes:

Best Human Practice Advance:

We feel that we deserve this prize for three reasons:

We explored the impacts, *both positive and negative*, of synthetic biology as a solution to real world problems, through interviewing professionals working in a relevant field, namely the impact of arsenic water contamination in Bangladesh.

We recognized existing problems with the way the current direction of synthetic. On going through the registry we found that most of the characterization data for biosensing parts is often neither comparable nor replicable. We have worked to solve this issue, for example with our ratiometric dual channel output.

*Our project doesn’t stop here*, in Chanel number 6 (Team:Cambridge/HumanPractices/FutureDirections) we considered the future implications and technological applications of our project, as well as the means by which it could be improved by subsequent users. We feel that the end to an iGEM project should not be the conclusion of an idea, but the start of it.

Best BioBrick Measurement Approach:

It is absolutely vital that a quantitative, numerical, robust, and flexible measurement approach exists to relay information to a user that is an accurate representation of the input processed by a biological device. Working from these principles, the following was done:

We designed and built Biologger, a *cheap, arduino-based, fully functional automatic rotary device* that has an incorporated ratiolumnometer

Our project is entirely open-sourced and open-platform. We have published source code for the two applications which serve to operate the device, one for PCs and the other for Android devices, as well as the open source circuit design that provides this ratiometric reading. Furthermore, the Android app is able to receive its data wirelessly, which we feel is a great advance in BioBrick measurement.

Our dual-channel luciferase reporter was successfully tested with a dilution series of E.coli transformed with the Lux Operon (under pBAD) biobrick (Part BBa_K325909) of the Cambridge iGEM 2010 team. It can detect, with good accuracy, both different light intensities, as well as the percentages of blue or orange frequencies in a sample.

Our device was successfully tested using artificial light to detect different frequencies (colours) as well.

Having done all the above, we believe that this fully open-sourced instrumentation kit (mechanical) chassis, electronics, software code), estimated at *$35.00* (or $85.00 if a Bluetooth modem is required), is a complete BioBrick measurement solution for any and all BioBricks with a light output.

Please explain briefly why the judges should examine these other parts:

Magnesium Sensitive Riboswitch BBa_K911001As a riboswitch sensing construct, this part is an entirely new type of biosensor (along with the fluoride construct) that could potentially change the way we think about designing input genetic circuits. Unlike the fluoride riboswitch, it is a derepression system and therefore serves to demonstrate the principle that riboswitches can be used regardless of whether they turn on or off their reporter.

Fluorescent ratiometric construct for standardizing promoter output BBa_K911009Fluorescence is a major cornerstone for biosensors in the registry, however, most parts do not involve the use of a ratiometric output, which has been shown in the literature to provide much more reliable and meaningful data. This part not only furthers the development of ratiometric measurements in molecular biology but due to the choice of promoters and terminators it can be used to characterize the difference in activity between E. coli and B. Subtilis

Fast Germination (B. subtilis) BBa_K911008This part is entirely novel for the registry and fully utilizes the recombination machinery inherent in the Bacillus chassis. Have spores that can germinate at a faster rate is certainly a worthy achievement and could help with experiments with B. Subtilis that any future iGEM teams may wish to perform.

Colony PCR:

Used to amplify DNA directly from cell culture, the reaction is the same as for normal PCR but with modified reaction composition and cycle settings, here shown for a 50 µl reaction using Taq polymerase.

This technique should only really be used for crude PCR assays, such as diagnostic PCR. If high quality DNA is desired, Miniprep followed by standard PCR should be used.

If taking a colony from a plate, resuspend the cells in ~50μl of water. Take 1 μl of this resuspension and use it as your template. Failing to do so will result in you having too much genomic DNA in your reaction, which will interfere with gel electrophoresis - you can tell if this has happened if a bright band appears where your wells are on the gel.

Resuspended cells can be stored in the fridge for a couple of days for eventual plating, if colony PCR proves successful.